Discussion
Drought stress is a leading constraint affecting grain yield and quality in soybean (Fried et al. , 2019). Genetic basis and mechanisms underlying drought tolerance at germination stage has not been extensively studied in soybean. Tolerance to drought stress is very complex; hence, elucidation of molecular mechanism regulating drought tolerance has been the long-term interest of soybean breeders. Identification, cloning and exploitation of stress responsive genes using molecular breeding/transgenic techniques is essential to develop drought tolerant soybean cultivars. Although, linkage and association mapping has identified genomic locus underlying drought tolerance (Semagn et al. , 2013; Wang and Qin, 2017), but attempts to understand drought tolerance at the transcriptome level was limited especially at germination stage. In this context, the present study used RNA-seq analysis to explore the global transcriptome of four contrasting genotypes viz., two tolerant (DTP & DTL) and sensitive (DSP & DSL) genotypes under drought stress at germination stage. Our results revealed that tolerant genotypes (DTP & DTL) possess higher RL compared to sensitive genotypes under stress treatment. This can be explained by the fact that RSA are important sensors of drought tolerance, and increased root length and deep root system greatly increases the moisture absorption and nutrient extraction for plant survival under water deficit stress (Wasaya et al. , 2018).
Both Venn diagram and Circos analysis suggests that sensitive genotypes are comparatively more vulnerable to drought stress at the transcriptomic level (Fig 2A, B & C ). Similar findings are previously reported in several other crop plants under water-deficit and other stresses ( Muthusamy et al. , 2016; Fracasso et al. , 2016). These results can be explained by the fact that compared to tolerant genotypes, sensitive genotypes reveal dramatic changes in morpho-physiological and biochemical parameters while mitigating negative impact of drought stress (Yang et al. , 2017). The higher correlation of DTP with DTL compared to DSP and DSL provides explanation about increased drought tolerance in DTL, because genes/alleles for drought tolerance in DTL are derived from wild-parent/DTP, while same genes/alleles for DSL comes from DSP (Fig. 3 ).
Both GO and MapMan analyses indicated that drought stress response was highly enriched in the DEGs associated with water transport, cell wall, plant-type vacuole membrane, antioxidant activity, catalytic activity, auxin transport, peroxidase activity, protein kinase activity, TF activity, secondary metabolite synthesis, hormone signaling and signaling (Table 4 ). Under water deficit conditions, plant cells must maintain functional integrity and rapidly remodeled to keep cell wall flexible under abiotic stress (Houston et al. , 2016). In this context, cell-wall remodeling enzymes such as pectin esterases (PME), expansins, xyloglucan endotransglucosylase/hydrolase (XTH) as well as glycine-rich cell wall structural protein (GRP) are involved in maintaining cell wall rigidity, and permits to cope with water deficit conditions (Tenhaken, 2015). However, most of these cell wall remodeling enzymes were highly expressed in tolerant genotypes but down-regulated in sensitive ones, indicating their vital role in cell wall remodeling under drought stress (Table 4 ). For instance, Arabidopsis lines overexpressing PMEI1 (pectin methylesterases) exhibited improved germination rate and seedling root growth under water deficit condition (An et al. 2008). Cho et al. , (2006) described that CaXTH3overexpressing-plants in Arabidopsis showed an increase in drought tolerance, and XTHs have been reported to be differentially regulated in maize under drought stress (Zhu et al. , 2007). Furthermore, overexpression of expansin gene (TaEXPB23 ) improves drought tolerance in tobacco. Similarly,AtEXP2  is involved in seed germination and drought stress response in Arabidopsis (Yan et al. , 2014). Yang et al. , (2014) also reported that overexpression of AtGRP2and AtGRP7 significantly influences drought tolerance in transgenic rice, and revealed that rice plants overexpressingGRPs were more tolerant to water deficit relative to wild-type plants. Xuan et al. , (2010) also demonstrated role ofNtGRP-1a  transcripts under drought stress in tobacco. Hence, up-regulation of above cell wall related genes might play essential role in seed germination and drought tolerance.
Under stress conditions different secondary metabolites i.e., lipids, amino acid and carbohydrate are accumulated in higher plants (Akula and Ravishankar, 2011). Shikimate pathway not only act as connection between central and secondary metabolism but also serve as precursor for most of the other secondary metabolites (Fig. S1 ; Maeda and Dudareva, 2012). Biosynthesis of tyrosine and phenylalanine through the shikimate pathway leading to the synthesis of wide range of secondary metabolites (Less and Galili, 2008; Gill and Tuteja, 2010). Isoquinoline alkaloids  are derived from  tyrosine, while indole alkaloids are produced by metabolic engineering of the tryptophan preventing plants from oxidative stress (Figure. S1 ; Sato and Kumagai, 2013). Phenylalanine acts as a precursor for diverse secondary metabolites and phenylalanine ammonia-lyase (PAL) takes part in phenylpropanoid biosynthesis; an essential step towards biosynthesis of anthocyanin’s, flavonoids, stilbenes, lignins and other compounds (Deng and Lu, 2017). It is reported that lignin and anthocyanin’s biosynthesis related genes were highly expressed under water stress in rice roots (Yang et al. , 2006). In our findings, many unique genes related to biosynthesis of lignin, alkaloids, flavonoids and anthocyanin’s such as PAL, STR, and laccase were highly expressed in tolerant genotypes, but mostly down-regulated in sensitive genotypes under drought (Table 4 ), proposing their diverse role towards drought tolerance. Among all the DEGs involved in the secondary metabolite biosynthesis, highest number of genes associated with lignin biosynthesis especially the laccase (LAC) and cinnamoyl-CoA reductase (CCR) genes indicating essential role of lignin in drought tolerance. Lignin reduces cell wall water penetration and transpiration in plants, and also maintain cell osmotic balance and membrane integrity under drought stress (Liu et al ., 2018). Moreover, laccase genes participate in the oxidation of flavonoids, and plays fundamental role in plant defense responses (Turlapati et al. , 2011). Flavonoids serve as antioxidant, and provide shield to plants against abiotic stresses (Pourcel et al. , 2007). For example, AtLAC2 inArabidopsis is involved in drought stress tolerance (Cai et al. , 2006). Liu et al. , (2017) reported accumulation of terpenoid indole alkaloids (TIAs) in the C. roseus  under drought stress. Hence, considerable upregulation of DEGs related to secondary metabolite biosynthesis in drought-tolerant genotypes, suggesting their vital role in drought tolerance.
Drought stress enhances reactive oxygen species (ROS) accumulation resulting in cell wall degradation and membrane damage.(Helena and Carvalho, 2008). Studies have demonstrated that ROS accumulation impaired seed germination in different crops including soybean (Ishibashi et al. , 2013). In this regard, plant utilize antioxidant defense system for scavenging ROS to avoid oxidative stress, and allows proper seed germination (Xie et al. , 2019). In our data, many redox related DEGs including GST, POD, SOD, glutaredoxin and thioredoxin were highly expressed in the drought-tolerant genotypes (DTP and DTL), whereas the same genes were down-regulated in drought-sensitive genotypes viz., DSP and DSL (Table 4 ). Many studies have revealed induced expression of GST under various abiotic stresses, including drought (Kumar and Trivedi, 2018). For instance, Bhardwaj and Yadav, (2012) reported an increase in expression of GST, POD, CAT, and SOD in horsegram subjected to drought stress, indicating their important role in drought stress tolerance. In addition, over-expression of AgAPX1  (ascorbate peroxidase) in Arabidopsis led to increase in ascorbate content and drought tolerance (Liu et al. , 2019). A glutaredoxin gene viz., SlGRX1 exhibited tolerance to oxidative and drought stresses in tomato (Guo et al. , 2010). Similarly, NADPH-dependent thioredoxin reductase A (NTRA ) mutant exhibited tolerance to drought and oxidative stress (Cha et al . 2014).
In soybean and other crop species, many transcriptome studies has revealed involvement of several TFs including, MYB, ERF/DREB, bHLH, NAC and WRKY in seed-germination process under stress conditions (Leeet al. , 2015; Raineri et al. , 2016; Baillo et al. , 2019). Members of these TF families may have either positive and negative regulatory role in the drought stress tolerance. The MYB are most abundant among all TFs expressed in present study, and many of these TFs are differentially regulated in both tolerant and sensitive genotypes (Table 3 ), suggesting their essential roles in germination in response to drought stress. For example, OsMYB6overexpressing plants in rice enhanced tolerance to drought stress (Tanget al. , 2019). Similarly, Zhao et al. , (2018) reported the involvement of TaMYB  towards drought tolerance in Arabidopsis . Overexpression of the ZmMYB3R andScMYBAS1 has been reported to enhance drought stress tolerance in maize (Wu et al. , 2019) and rice (Peixoto-junior et al. , 2018), respectively. After MYB, the bHLH followed by ERF, NAC and WRKY represent the highest number of differentially expressed TFs under drought stress. For example, bHLH family member ZmPTF1 andVvbHLH1 regulates drought tolerance in maize (Li et al. , 2019) and Arabidopsis thaliana (Wang, et al. , 2016), respectively by promoting root development, ABA synthesis and accumulation of flavonoids. The AP2/ERF TF were revealed to modulate brassinosteroid-regulated plant development and drought responses inArabidopsis (Xie et al. , 2019). Moreover, NAC genes  such as JUNGBRUNNEN1 in tomato (Thirumalaikumar et al. , 2018) and OoNAC72 Arabidopsis (Guan et al. , 2019), positively regulates drought tolerance, whereas, SbNAC052 , SbNAC073 , and SbNAC116 serve as negative regulator in drought stress tolerance in sorghum (Sanjari et al. , 2019). Many TFs from WRKY family exhibited function in drought tolerance in various crops, for example overexpression of GmWRKY12 in soybean (Shi et al.2018 ), TaWRKY2 and AtWRKY30 in wheat (Gao et al. , 2018; El-Esawi et al. 2019), and ZmWRKY40 in Arabidopsis (Wang et al.2018 ) has led to enhanced drought tolerance in these crop plants. Hence, these TFs can be an important target for breeding drought tolerance in soybean.
In the present study, many genes related to plant-specific Ca2+ signaling such as calcium-binding proteins, calcium ATPases, calmodulin-like proteins (CMLs), calmodulin-binding protein, calmodulin-binding receptor, Ca2+-dependent protein kinases (CPKs), and Annexin were mostly up-regulated in tolerant genotypes as compared to sensitive genotypes. (Table 4 ). For example, Campo et al. , (2014) reported that OsCPK4overexpression in rice showed an increase in drought tolerance by reducing lipid peroxidation and electrolyte leakage.  Plasma membrane Ca2+-ATPase directly regulates drought stress tolerance by activating ABA signaling pathway (Shao et al. , 2008; Cerana et al. , 2006) and the increased ABA accumulation leads to stomatal closure and expression of many stress-related genes. For example, Cerana et al ., (2006) reported overexpression of Ca2+-ATPase viz.,ACA8  and ACA9  stimulated ABA accumulation. Hence, it has been proposed that Ca2+-ATPases might play important role in drought stress response through ABA signaling, as the latter is well-established mediator of drought stress adaptation in plants (Qudeimat et al. , 2008). Calmodulin-like Proteins such asCML20 in Arabidopsis (Wu et al. , 2017),ShCML44 from Solanum habrochaites (Munir et al. , 2016)  were revealed to induce drought tolerance by regulating ABA signaling in guard cells. The annexin gene AnnSp2 exhibited drought tolerance in overexpressed transgenic tomato plants through ROS-scavenging and modulation of ABA synthesis (Ijaz et al. , 2017). Wei et al., (2014) reported that OsCPK9,calcium-dependent protein kinase in rice is involved in tolerance to drought stress. Hence, the above findings suggest close relationship between ABA and Ca-signaling in drought stress response. Evidence indicate that Ca2+/CaM is involved in ABA-induced drought signaling under PEG stress, and ABA-synthesis was associated with cytoplasmic Ca2+ concentrations (Li et al . 2002). As reported previously, ABA activates cytosolic Ca2+ in guard cells by maintaining turgor within guard cells that leads to stomatal closer and prevention of transpiration water loss, and ultimately induces drought tolerance (Song et al. , 2008). Furthermore, receptor-like protein kinase (RLKs) and mitogen-activated protein kinases (MAPKs) are key components for signaling pathways in plant and have diverse function in seed germination by regulating stress-responsive gene ( Baek et al. , 2019; Jagodzik et al. , 2018). For instance, seed germination was significantly enhanced by expression of SpMAPK3 andSlMAPK3 in response to abiotic stresses (Muhammad et al. , 2019; Li et al. , 2014). The MAPK kinase10.2 promotes drought tolerance by activating different MAPKs in rice (Maet al. , 2017). Cysteine-rich RLKs such as CRK45 inArabidopsis (Zhang et al. , 2013) and TaCRK41 in wheat (Chen et al. , 2017) involved in ABA signaling and positively regulates seeds germination under drought and oxidative stress. Similarly, in Arabidopsis, proline-rich RLKs, PnLRRRLK27exhibited tolerance towards abiotic stress during seed germination (Wanget al. , 2017). Consistent with these results, we also observed up-regulation of MAPKs and RLKs in tolerant genotypes, whereas, reverse trend was seen in sensitive genotypes. Therefore, the above results propose that MAPKs, RLKs and Ca2+ signaling together with ABA might play key role to regulate seed germination under water deficit conditions.
Numerous transgenic studies have demonstrated that overexpression of Aquaporins (AQPs) viz., TIPs and PIPs enhanced drought tolerance probably by endorsing stomatal closure and regulating the plant hydraulics (Zargar et al. , 2017). For example, VfPIP1overexpressing plants in Arabidopsis thaliana exhibits drought tolerance through promoting stomatal closure (Cui et al. , 2008). Pou et al. , (2013) reported the putative role of PIP andTIP genes in leaf hydraulic and stomatal conductance in grapevine under drought stress. Although role of AQPs in dehydration tolerance has been extensively studied in plants but still conflict of interest remained among the researchers regarding their up- or down-regulation during water deficit condition. Under drought stress, considerable variation was observed in the expression of PIPs at the transcript level; for example, significant upregulation of somePIPs was observed while expression of others remained either unchanged or downregulated (Lian et al. , 2006; Guo et al. , 2006). Upregulation of some AQPs might assist in maintaining the normal physiological processes in plant and resist the stress, while other may help to adapt or tolerate the stress condition by reducing their own activity and expression (Zargar et al . 2017). Hence, in the present study some unique AQP genes are differentially regulated in tolerant and sensitive genotypes proposing their innate role in drought tolerance (Table 4 ).
Phytohormone signaling is complex and plays important regulatory role in drought responsive pathways of soybean (Pandey et al , 2017). In recent study, genes associated with phytohormone biosynthesis and signaling viz., AUX, ETH, ABA and GA represent most number of DEGs in both tolerant and sensitive genotypes under drought stress (Table 2; Table 4 ). For example, auxin/indole-3-acetic acid (Aux/IAA), small auxin‐up RNA (SAUR), indole-3-acetic acid-amido synthetase/Gretchen Hagen (GH3) and auxin efflux carrier component (PIN) showed dynamic changes in the roots of tolerant and sensitive genotypes under drought stress; however, these genes are highly expressed in tolerant genotypes, whereas, reverse trend was observed in sensitive genotypes, indicating complex role of auxin signaling. Zhang et al. (2012) revealed that auxin efflux carrier component such asOsPIN3t in rice involved in auxin transport and response to water stress. The auxin-sensitive Aux/IAA proteins has been revealed to mediate drought tolerance through regulating glucosinolate levels in Arabidopsis (Salehin et al. , 2019). Under drought stress, overexpression of auxin-responsive protein, TaSAUR75 exhibited an increase in root length and survival rate in Arabidopsis(Guo et al. , 2018). Similarly, GH3 genes of Chickpea (CaGH3-1 & CaGH3-7 ) and Medicago truncatula(MtGH3-7MtGH3-8  and MtGH3-9 )  were highly induced under drought stresses (Singh et al. , 2015). In our findings, many GH3 genes were highly induced in tolerant genotypes, respectively, suggesting their important role in stress adaptation. Ethylene biosynthesis mainly involved two enzymes viz., aminocyclopropane-1-carboxylic acid (ACC) oxidase and ACC-synthase (ACS) in various crops (Yoon and Kieber, 2013; Van Der Straeten et al ., 2001), and this hormone regulates seed germination and seedling growth under abiotic stresses (Huang et al. , 2019). Similar with these findings, our study also exhibited higher expression levels of ACS and ACO in tolerant genotypes. In addition, it is also reported that hormonal balance between ABA and GAs is necessary to regulate seed germination (Miransari and Smith, 2014). In present study, DEGs associated with gibberellin 2-beta-dioxygenase, Gibberellin 3-beta-dioxygenase, Gibberellin 20 oxidase, and DELLA protein were highly expressed in tolerant genotypes as compared to sensitive genotypes (Table 1). Habib et al. , (2019) demonstrated that SlGRAS7  TF improved drought tolerance by enhancing gibberellin/auxin signaling. Consistent with a recent report, we also identified many enzymes related to ABA biosynthesis such as 9‐cis‐epoxycarotenoid dioxygenase (NCED), abscisic acid 8′-hydroxylase and carotenoid cleavage dioxygenases (CCD). These enzymes were differentially regulated in tolerant and sensitive genotypes resulting in better hormonal balance under drought stress (Shu et al. , 2018). In plants, ABA plays diverse role in response to drought as well as in various developmental processes such as seed germination. Drought stress enhances plant ABA accumulation resulting in ABA-receptor complex (PYR/PYL/RCAR–PP2C–ABA) that triggers SnRK2 protein kinases, and this kinase facilitate stomatal closure and reducing transpiration water loss, and maintaining water balance within plant under water-deficit condition (Fujita et al. , 2013). Brassinosteroids (BRs) are plant steroid hormones, which regulate the expression of stress related genes. Brassinosteroid Insensitive 1 (BRI1) was highly expressed in tolerant genotype, and previous study has revealed that BRI1 plays essential role in plant growth, development and drought tolerance. For example, Feng et al. , (2015) using RNAi suggested that mutation of bdBRI1 in Brachypodium distachyon resulting in dwarf phenotype with enhanced tolerance towards drought stress.
Based on the findings of GO enrichment, PPI network and dChiP analysis as well as screening of genes within previously reported genomic/hotspot regions associated with drought tolerance in soybean on Chr.08, a total of ten genes were considered as the most possible candidate genes regulating drought tolerance at germination stage in soybean (Table 5 ). Function annotation revealed thatGlyma08G162700 function as peroxidase 3 (POD 3),Glyma08G091400 as ‘glutamate decarboxylase 1-like’,Glyma08G097300 as peroxisomal (S)-2-hydroxy-acid oxidase GLO1-like , Glyma08G045000 as Ca-transporting ATPase,Glyma08G271600 function as glycine-rich cell wall structural protein, Glyma08G014200 function as tubulin beta-1 chain-like,Glyma.08G201700 function is uncharacterized; the remaining three predicted genes viz., Glyma08G042100 , Glyma.08G029400 andGlyma.10G184500 function as MYB184, MYB127 andMYB78, respectively (Table 5) . The POD 3 enzymes were reported to participate in plant development, stress responses and hormone signaling (Wu et al. , 2019); for example, AtPrx3participates in positive regulation drought stresses response in Arabidopsis (Llorente et al. , 2002). Yong et al . (2017) reported that inhibition of glutamate decarboxylase activity result in the increase of endogenous glutamate (Glu), and that in turn enhanced drought tolerance in white clover. Down-regulation of glycolate oxidase pathway reduces peroxisomal H2O2 production in the green tissues of plants under drought stress, and provides tolerance to oxidative stress (Zhou et al ., 2007; Noctor et al ., 2014). The Ca2+ATPase such as OsACA6 confers drought stress tolerance with reduced accumulation of ROS and enhanced the expression of stress‐responsive genes in tobacco (Huda et al. , 2013). Involvement of Ca2+-ATPase in drought tolerance has been revealed through transcript profiling of a sweet potato (Yanget al. , 2018). Moreover, in B. napus , two cell-wall related proteins viz., glycine-rich and fasciclin-like arabinogalactan were induced under drought stress (Koh et al. , 2015). Panet al . (2018) reported highly induced expression of tubulin beta-1 chain-like in foxtail millet under drought stress (Pan et al. , 2018). The tubulin beta-1 (R83) chains are major constituents of microtubules and their accumulation also peaked at 48 h after the onset of drought stress (Bian et al. , 2017). In addition, many of the earlier studies has explained the important role of MYB gene family in the drought stress tolerance in crop plants (Tang et al ., 2019; Zhao et al ., 2018; Wu et al ., 2019; Zhang et al ., 2019; Peixoto-Junior et al ., 2018). Hence, the above ten genes that includes three hub genes were predicted as the most possible candidate genes regulating drought tolerance at germination stage in soybean. Therefore, these genes required further validation to prove their actual role and use in soybean improvement.
Based on the above findings, a hypothetical model was proposed as shown in Fig. 11 . This comparative model explains, how aquaporin’s, cell-wall related enzymes, secondary metabolites, antioxidants, kinases, MAPK signaling and TF activities functions in drought response between drought tolerant and sensitive genotypes.